Fig. 3. All three cis-regulatory elements of the Eklf locus contain highly conserved Smad binding motifs adjacent to Gata sites. (A) Alignment of five mammalian Eklf genomic loci encompassing 17 kb (30 kb global alignment length) surrounding the Eklf transcription unit in between the two nearest neighboring genes, Gcdh and Dnase2a. Each sequence is represented as a black line (breaks demarcate gaps in the alignment), repeats or low complexity DNA are represented as blue bars, untranslated regions (UTR) as light tan bars and exons as brown bars. The degree of sequence conservation between species is expressed as an alignment score (y-axis) per nucleotide position (x-axis; global length). Peaks of conservation above 0.6 (red line) within non-coding regions are shaded in orange. The 950 bp region upstream of Eklf exon 1, exon 1 itself, and intron 1 are highlighted in light yellow. (B) Enlarged view of the aligned mouse Eklf sequence from -950 bp to the beginning of exon 2. The three conserved Eklf cis-regulatory elements are indicated (upstream enhancer, proximal promoter, intronic enhancer) together with blocks of four or more nucleotides of perfect homology (green bars), erythroid hypersensitive sites (EHS1 and EHS2, dark yellow bars) and previously known Gata and Cp1 sites (small red bars). Arrows indicate positions of newly identified Smad binding motifs (SBM) and WGATAR motifs. (C) Detail of seven-species alignment of all three Eklf cis-regulatory regions (upstream enhancer, proximal promoter, intronic enhancer) at the single nucleotide level. Newly identified Smad binding motifs (SBM1 through SBM10) are highlighted (blue), as are unassigned conserved blocks of perfect homology (green), WGATAR motifs (red), E-box motifs (yellow), Cp1 (pink) and putative Sp1 (brown) sites. The EHS1/GEG region within the upstream enhancer is underlined.